The pathology of pulmonary disorders due to Aspergillus spp.
Abstract: * Diagnosing the range of pulmonary disorders caused by Aspergillus spp can be challenging. In instances of hypersensitivity responses to Aspergillus spp (ie, allergic bronchopulmonary aspergillosis, bronchocentric granulomatosis, and hypersensitivity pneumonitis), the surgical pathologist must be cognizant of their relationship to infection, as fungal organisms may be rare or absent from the biopsy specimens. Within the spectrum of opportunistic infection, it is critical to distinguish Aspergillus spp from other fungal mimics, as well as to discern whether infection is limited, progressive, or immanently life threatening. However, the surgical pathologist who establishes expertise in this area will be rewarded by the satisfaction of having contributed primarily to an important area of patient care. This article reviews the spectrum of pulmonary disorders due to infection by Aspergillus spp, with emphasis on the clinical implications of diagnosis.

(Arch Pathol Lab Med. 2008;132:606-614)
Subject: Mycoses
Bacterial pneumonia
Pneumonia
Authors: Kradin, Richard L.
Mark, Eugene J.
Pub Date: 04/01/2008
Publication: Name: Archives of Pathology & Laboratory Medicine Publisher: College of American Pathologists Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2008 College of American Pathologists ISSN: 1543-2165
Issue: Date: April, 2008 Source Volume: 132 Source Issue: 4
Accession Number: 230151947
Full Text: Aspergillus spp are the cause of a variety of pulmonary abnormalities that range in severity from commensal overgrowth of airways to invasion of the lung and its blood vessels, leading to sepsis and death. The spectrum of disorders attributable to Aspergillus spp (Figure 1) requires surgical pathologists to be acquainted with both their diagnostic features and clinical implications. (1) Advances in the development of antifungal antibiotics demand that Aspergillus spp be distinguished from other hyphal fungi that can mimic their morphologic features. In addition, as confirmatory microbial cultures may not be available for all cases, establishing expertise in discerning the morphologic features of Aspergillus spp and the spectrum of histopathologies caused by them is critical in directing appropriate therapy

There are approximately 300 species of the genus Aspergillus. They are ubiquitous within the environment, but only approximately 8 species are responsible for the vast majority of human disease. (2) Aspergillus fumigatus is the most common pathogen, followed by A niger, with A nidulans, A terreus, A clavatus, Aflavus, A niveus, and A ustus accounting for most other infections

Aspergillus spp are readily isolated from both soil and decaying vegetation, but they have been isolated from a wide range of organic substances, including foodstuffs, paint, medications, refrigerator walls, dialysis bags, etc. The fungal spores of Aspergillus spp are released into the ambient air, and viable organisms can survive extreme climactic conditions (eg, Antarctica, the Saharan desert, and high altitudes). Additionally, Aspergillus spp can survive in extremes of acid-base balance.

MICROSCOPIC MORPHOLOGY

Aspergillus spp are reliably demonstrated in tissue sections with silver histochemical stains (eg, Gridley or Gomori methenamine-silver). (3) These decorate the fungal wall gray-black. The hyphae of Aspergillus spp range in diameter from 2.5 to 4.5 [Am and exhibit frequent septation. When septae are not apparent, hyphae may be mistaken for those of Zygomyces spp. Aspergillus spp hyphae tend to branch dichotomously, progressively, and primarily at acute angles of approximately 45[degrees], mimicking an arborizing tree branch (Figure 2). When cut in cross sections, hyphae can be mistaken for yeast or spores; however, the absence of budding suggests their true nature (Figure 3). In areas of mycelial growth, hyphae often become tangled, bulbous, and distorted, so that it may be difficult to confirm the diagnosis with accuracy based exclusively on morphologic grounds (Figure 4).

The aspergillum, a ritual liturgical implement used in Roman Catholic ceremonies, resembles the fruiting body of Aspergillus spp, and it is from this implement that these fungi get their name (Figure 5). Fruiting bodies develop from mycelia in areas of high oxygen tension, such as lung/or sinus cavities, and do not develop in tissues. The fruiting body is composed of a vesicle and either 1 or 2 layers of phialides that produce conidia, and the morphology of the fruiting body may allow Aspergillus spp to be accurately speciated in situ.

INFLAMMATORY RESPONSE TO ASPERGILLUS

Aspergillus spp produce a variety of virulence factors, including endotoxins, heparin-like factors, and oxalic acid. The hyphae of Aspergillus spp evoke histologic responses by the host that reflect the level of immunocompetence. In chronic infections, hyphae often show evidence of coating by plasma proteins, including immunoglobulin and complement. This can range in extent from individualized sheaths around hyphae (Figure 6) to extensive amorphous hypereosinophilic coatings around mycelia, a phenomenon termed the Splendore-Hoeppli phenomenon (Figure 7).

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Invasive Aspergillus infections are characterized by tissue necrosis accompanied by neutrophilic exudates. Eosinophils are variably present. Macrophages are invariably present, and granulomatous inflammation with giant cell formation is common (Figure 8). Nonnecrotizing sarcoidal granulomas may develop in the walls of noninvasive fungus balls.

IMMUNOLOGIC DISORDERS DUE TO ASPERGILLUS INFECTION

Allergic bronchopulmonary aspergillosis (ABPA) includes chronic reversible airways disease (asthma) and bronchiectasis with noninvasive colonization of the airways by Aspergillus spp. (4) It is not certain whether the fungi play an opportunistic role in exacerbating atopic responses or are primary in the pathogenesis of this disorder. (5) Patients develop intractable bronchospasm, with blood eosinophilia and elevated serum immunoglobulin E levels. The disorder is characterized by central cystic bronchiectasis, with mucoid impaction of the proximal airways of the upper lung. (6) The impacted mucus is viscid and may form a cast of the airways, a disorder termed plastic bronchitis. Microscopically, the mucus plugs show layers of degenerating eosinophils interspersed within the mucin (Figure 9). In addition, the lung may show patchy peribronchiolar eosinophilic pneumonia.

Most cases of ABPA are diagnosed clinically and do not come to biopsy. However, the disease may be diagnosed following the microscopic examination of an expectorated mucus plug showing allergic mucus and fragmented fungal hyphae, or in lungs surgically resected for other diagnoses.

Fragmented fungal hyphae can be difficult to identify in ABPA, and silver stains should be applied routinely to the evaluation of allergic mucus plugs. Whereas the clinical and histologic features of this disorder are most frequently caused by Aspergillus spp, other fungi (eg, Candida spp) can yield comparable changes. A subset of patients with cystic fibrosis may develop concomitant ABPA, and establishing the diagnosis in this setting can be difficult. (7)

BRONCHOCENTRIC GRANULOMATOSIS

Bronchocentric granulomatosis (BCG) reflects an abnormal cell-mediated response to Aspergillus spp in which small-caliber airways develop circumferential granulomatous inflammation, loss of the normal lining respiratory epithelium, and impaction of the airway lumen by a granular basophilic mucin and cellular debris (Figure 10).6 The disorder may be first noted radiographically as isolated or multiple airway-centered nodules. (8) At times, it may be difficult to distinguish radiographically from neoplasia. BCG may be part of the spectrum of ABPA or occur as an isolated disorder. As in ABPA, Aspergillus spp are often fragmented hyphae and difficult to identify. Surrounding areas of eosinophilic pneumonitis are common. When the disease is suspected clinically, it can be treated noninvasively with corticosteroids. However, definitive resection may be indicated in order to exclude neoplasia.

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HYPERSENSITIVITY PNEUMONITIS

Hypersensitivity pneumonitis (HP) reflects a combined abnormality of humoral and cell-mediated immunologic responses to organic antigens. Most cases of HP are caused by thermophilic actinomycetes, but hypersensitivity to Aspergillus spp is well documented. Sources include moldy hay, compost, esparto grass, home humidifiers, and malt (A clavatus). The pulmonary pathology of HP depends on whether the disease is acute, subacute, or chronic. Upper lobe predominance is the rule in HP, and it may be a helpful feature in establishing the diagnosis. Although the diagnosis is based primarily on establishing an historic link between antigen exposures and clinical findings, lung biopsies can be helpful, as certain histopathologic features strongly suggest HP, particularly in its subacute phase. The lung shows bronchiolocentric, lymphohistiocytic, interstitial infiltrates with poorly formed microgranulomas and giant cells that contain birefringent crystals (Figure 11). (9) Whereas CD8+ lymphocytes characteristically predominate in bronchoalveolar lavage fluid specimens, immunostains may reveal dominance of either [CD4.sup.+] or [CD8.sup.+] lymphocytes in situ. The presence of interstitial and alveolar eosinophils is characteristically seen in HP due to Aspergillus antigens but is rare in the response to other antigens

Whereas these pathologic features strongly correlate with the clinical diagnoses of HP, at times it may be difficult to establish antigenic causation. In addition, other histopathologies, including nonspecific cellular interstitial pneumonitis, obliterative bronchiolitis, lymphoid interstitial pneumonitis, and nonnecrotizing granulomatous inflammation resembling sarcoidosis, can be seen with HP.

The diagnosis is confirmed by careful documentation of patient history, a syndrome of repetitive episodes of pneumonia-like illnesses following exposures, and serologic testing for serum precipitins to the offending antigens. In chronic HP, the lung shows a pattern of interstitial fibrosis that mimics usual interstitial pneumonitis, except for the presence of rare microgranulomas. The prognosis of patients with chronic HP is guarded, and patients may show progressive lung scarring, leading to respiratory failure that is not responsive to corticosteroids or removal of the offending antigen.

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ASPERGILLUS BRONCHITIS AND CHRONIC NECROTIZING ASPERGILLOSIS

Bronchial and bronchiolar infections by Aspergillus spp are poorly recognized entities. They are generally seen in the setting of modest immunosuppression accompanying disorders like diabetes mellitus, or the use of aerosolized steroids in asthmatics. In many cases, they are precursor lesions for invasive disease. Fungal hyphae may be observed filling the lumen of airways without evidence of chronic commensal growth (eg, the Splendore-Hoeppli phenomenon) or frank tissue invasion. Elastic stains are helpful in determining whether organisms have begun to transgress normal tissue barriers (Figure 12).

FUNGUS BALLS

The colonization of old fibrocavitary disease (eg, areas of bronchiectasis due to healed tuberculosis or sarcoidosis (10)) or emphysematous bullae by Aspergillus spp is the cause of pulmonary aspergilloma formation. The presence on chest radiography of an intracavitary density that is positionally mobile strongly suggests the diagnosis. The term mycetoma accurately applies to soft tissue infections and should not be applied to intracavitary fungal mycelial growth. Patients may be asymptomatic or present with episodes of hemoptysis that can be massive, necessitating emergent bronchial arterial embolization of feeding vessels or definitive resection of the fungus ball.

The morphology of the hyphae in aspergillomas is often distorted, and at times it can be impossible to identify the septate acute angle branching hyphae of Aspergillus spp. Fungus balls show areas of alternating staining intensity that are evident on hematoxylin-eosin-stained sections (Figure 13), giving the impression of alternating zones of fungal growth. The Splendore-Hoeppli phenomenon is invariably seen (arrows). The walls of the cavity are often superficially ulcerated and lined with granulation tissue, granulomatous inflammation, or metaplastic squamous epithelium, depending on the activity of the lesion. The occasional presence of germinative fruiting bodies of Aspergillus spp with characteristic phialides and conidial forms allows definitive speciation.

When the fungus ball is caused by A niger infection, one may occasionally observe rapid expansion of cavitary disease due to vascular thrombosis induced by calcium oxalate crystal deposition, a disorder termed chronic pulmonary oxalosis (Figure 14, A and B). Oxalic acid is produced by a variety of Aspergillus spp but is most commonly a feature of A niger infection. (11) Diffusion of oxalate into surrounding blood vessels can be prothrombotic and lead to extensive ischemic necrosis. These patients may also show oxalate crystal deposition in the renal tubules. (12) Resection of the fungus ball is definitive treatment.

The walls of all "benign" fungus balls must be carefully examined in order to exclude chronic necrotizing aspergillosis, in which there is evidence of fungal invasion of adjacent lung parenchyma but no angioinvasion. The application of silver and elastic stains is helpful in excluding fungal invasion. In addition, it is important to distinguish fungus balls that may form in areas of active necrotizing cavitary disease due to angioinvasive infection from benign intracavitary disease.

ANGIOINVASIVE ASPERGILLOSIS

This life-threatening infection is seen in patients who have been chronically immunosuppressed and/or have neutropenia. (13) It is a feared complication of bone marrow and solid organ transplantation, as well as antileukemic chemotherapies. Interestingly, angioinvasive aspergillosis is an uncommon complication of human immunodeficiency virus-related illness, despite profound immune deficiency. (14) The lung shows a necrotizing pneumonia with areas of hemorrhage and acute and granulomatous inflammation (Figure 15). Grossly targetoid lesions with central thrombosed vessels secondary to angioinvasion surrounded by a rim of consolidated lung (Figure 16), confluent bronchopneumonia, or dense lobar consolidation may be seen. Identification of fungal angioinvasion is enhanced with the aid of silver and elastic stains. Rarely, foci of infarcted lung yield an infected pulmonary sequestrum (Figure 17).

The fungal hyphae have a propensity to invade blood vessels and to "metastasize" to other organs. The presence of sunburst vasculocentric hyphal growth is diagnostic of "metastatic" foci of infection (Figure 18). Virtually any organ may be secondarily involved following vascular invasion within the lung. The thyroid is frequently involved at autopsy, but it is generally clinically silent premortem. Aspergillus spp can seed abnormal cardiac valves to produce endocarditis, and dissemination to brain may lead to fatal hemorrhagic infarctions.

DIAGNOSTIC AIDS

The gold standard for the diagnosis of Aspergillus infection is isolation and culture of the organisms in the microbiology laboratory. However, certain Aspergillus spp show characteristic morphologies. Aspergillus terreus is an opportunistic fungus that frequently infects patients with chronic granulomatous disease, (15) as well as other immunocompromised hosts. (16) Its hyphae are characteristically fragmented and pyriform and show orthogonal branches (Figure 19). The presence of pigmented conidia and oxalate crystals suggests infection by A niger (Figure 20). Fungus balls due to A nidulans have a propensity to produce pale-staining, thick-walled, nonviable Hulle cells that show Maltese cross birefringence when examined under polarized light (Figure 21, A and B).

Although the majority of acute angle branching septate hyphae encountered in medical practice are Aspergillus spp, the exceptions can be difficult to distinguish by conventional light microscopy and histochemical staining. Organisms that mimic Aspergillus include the Zygomyces, Pseudallescheria boydii (Scedosporium), and Fusarium spp. The Zygomyces are broad and pauciseptate and show predominantly orthogonal right angle branching (Figure 22), although acute angle branching is not uncommon and does not exclude the diagnosis. The hyphae of the Zygomyces are particularly well stained by standard hematoxylin-eosin. All hyphate fungi, including the Zygomyces, can produce fungus balls in the lung. Invasive infection by Zygomyces invariably requires both antibiotics and surgical resection

The hyphae of Pseudoallescheria are generally smaller than those of Aspergillus spp. The fungus branches predominantly at acute angles, but it tends to branch haphazardly rather than progressively. Terminal chlamydospores mimicking the appearance of a "tennis racket" may be frequent (Figure 23). The conidia produced by Pseudoallescheria in fungus balls are ovoid and pigmented. The septate hyphae of Fusarium spp branch irregularly, generally at right angles, and show constrictions at branch points (Figure 24). Whereas distinguishing these pathogens from one another in tissue may be exceedingly difficult, it is important, as the efficacy of available fungal antibiotics is dependent on diagnosis. Immunohistochemical reagents are available that may assist in the identification of Aspergillus spp (Figure 25). Currently available polymerase chain reaction techniques used on tissue sections have not generally proven reliable. Whereas both Aspergillus spp and Pseudallescheria are sensitive to voriconazole, Pseudallescheria is resistant to amphotericin. The sensitivities of Fusarium spp to most antifungal antibiotics are ill-defined but appear to be limited.

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CONCLUSION

Differentiating the effects of Aspergillus spp in lung biopsies can be challenging. However, as prognostic and therapeutic implications of diagnosis are often substantial, it is imperative that the surgical pathologist develop expertise in this endeavor. Whereas consultations with treating internists, surgeons, and radiologists can be of invaluable assistance in confirming histologic impressions, the treating physicians will often rely on the surgical pathologist to establish the final diagnosis in complicated cases.

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Richard L. Kradin, MD; Eugene J. Mark, MD

Accepted for publication September 12, 2007.

From the Departments of Pathology (Drs Kradin and Mark) and Pulmonary/Critical Care Medicine (Dr Kradin), Massachusetts General Hospital, Boston.

The authors have no relevant financial interest in the products or companies described in this article.

Presented in part at the 28th Annual Course, Current Concepts in Surgical Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, November 2006.

Reprints: Richard L. Kradin, MD, Department of Pathology, Warren 253, Massachusetts General Hospital, Boston, MA 02114 (e-mail: rkradin@partners.org).
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